There is a huge demand of new types of sensor surfaces and devices that can readily be functionalized for sensitive, selective and quantitative analysis of broad spectrum chemical and biological substances. It is desirable to study real-time molecular interactions, preferable in situ or even in vivo. This is important not only since this opens up the possibility for immediate signal read-out (high throughput), but also because it gives additional information of the kinetics and discrimination of multiple molecular binding. At the same time these sensor surfaces and devices should be suitable for repeated usage (mechanical and chemical stable), function in a wide range of environmental conditions (pH, temperature, pressure, and chemical environment), and yield reproducible results with minimum of recondition or sample preparation. Preferably, they should be made of materials that are amenable for easy and reproducible production, miniaturization, safe operation, high throughput, and ultimately biocompatible. For example, in disease diagnosis detection of multiple biomarkers is important in the diagnosis of complex diseases like cancer and neurological disorder. The use of devices capable of multiple markers detection in healthcare applications requires detection techniques with transducer materials that are selective, sensitive and biocompatible. Current transducer materials like polystyrene beads, carbon electrodes, gold, silicon, oxidized silicon and glass do not meet the requirements of smoothness, homogeneity, chemical and electrical stability, reproducibility, and biochemical surface modifications, and are not amenable for bio-integration [1].
There are a number of sensor devices and surface sensitive techniques available today which are capable of specific bio- and chemical sensing. The most common are sensitive to changes of mass (quartz crystal microbalance, QCM), refractive index (surface plasmon resonance, SPR), or fluorescent properties. [2-4] In general, all these techniques are based on the same detection principle, namely bonding of the analyte to specific (receptor) sites at the sensor surface. These methods can yield molecular information such as chemical identity, concentration, binding affinity, conformational properties, visco-elastic properties and thermodynamic parameters. Drawbacks with current commercial techniques include poor sensitivity toward small molecules (SPR and QCM), and unspecific analyte binding. Fluorescent techniques rely on changes of fluorescent properties, which work well for some molecules but not for others; otherwise tedious additions of fluorescent labels are required, which can interfere with molecular binding or make interpretation ambiguous.
A useful and surface sensitive optical technique is attenuated total reflection (ATR) spectroscopy. This is based on internal reflection in a material, or internal reflection element (IRE), with high refractive index, nIRE, which is much higher than the surrounding medium, typically nIRE>2 [5]. The electromagnetic waves that propagate inside the IRE produce an evanescent field across the interfaces to the surrounding media (with n<<nIRE) and may loose or gain energy by resonant excitation in the evanescent field region that penetrates into the adjacent low refractive index medium surrounding the IRE. The penetration length of the evanescent field, dp, depends on the angle of incidence (θ1) and the ratio of the refractive index of the waveguide and the surrounding medium. The ability of molecules in the immediate vicinity outside the IRE to absorb energy from the light propagating within the IRE by the evanescent field is the basis for all evanescence-wave spectroscopy (EWS) methods, which includes ATR spectroscopy. Fluorescent (TIRF), microwave, UV-vis, near-infrared, or mid-infrared (ATR-IR) spectroscopy may be performed in this way. A great advantage with EWS is that it can be used to study any molecule, independent of state of aggregation, size, charge, or fluorescent properties. In addition, it can give specific chemical interactions that unravel chemical interactions. The ATR-IR spectroscopy has been revolutionized by single or multiple reflection elements (MREs) combined with anvil-type pressing devices that allow virtually any type of samples to be analyzed with minimum sample preparation. In particular diamond is attractive. Apart from having the desired optical properties (broad band optical transmittivity and high refractive index; n=2.4), it has superior mechanical (large Young's modulus), thermal (high thermal conductivity) and chemical properties (it is chemical inert; it can operate in at all pH and temperature intervals of interest) that makes it the standard IRE material in most laboratories. ATR-IR has proved to be useful in a wide variety of applications, including chemical [6-8] and biological (protein, bacterial) identification [6; 9; 10], biosensors [2; 11; 12], catalysis [13], etc.
Diamond is also an attractive optical material for photonic and optoelectronic applications with advantageous broad band transmitting and intrinsic narrow band emission (e.g. due to N-V centers) properties. Developments in fabrication methods show that high-quality diamond can be produced and manipulated with great precision. The additional beneficial abovementioned physic-chemical properties thus make diamond a very promising material in future microelectronic and photonic application. Nanocrystalline diamond (NCD) or ultra-nanocrystalline diamond (UNCD) is a form of diamond where the grain size of crystallites is in the order of nanometer. Thin NCD films grown on Si substrates from methane-hydrogen gas mixture in a DC arc plasma CVD reactor yield optical transparency greater than 84% at λ>700 nm [14]. Surface roughness in the order of 5-50 nm for 1 μm thick films, significantly decrease the transmission in the visible because of light scattering, but has negligible effect in the IR range. NCD films are transparent in the IR and have optical constants n=2.34-2.36 and k=0.005-0.03. The micro-hardness is between 75-85 GPa, i.e. typical for diamond films. Diamond is an ideal coating in optical applications in harsh environments; it is chemically inert, strong, and broad band optically transparent. An important quality for a diamond optical coating is surface smoothness. NCD can be fabricated with great precision with excellent optical and mechanical properties that retains the attractive physico-chemical properties of diamond. A NCD surface is characterized by a large surface area due to the nanocrystalline structure. For the same reason the NCD surface contains a large number of low-coordinated carbon atoms that may form bonds to a large number of molecules. In fact, a major advantage with a NCD surface is that it can be manipulated in several ways to covalently bind a number of molecules (ligands) via e.g. amine, carboxyl and thiol coupling directly to or via linkers low-coordinated C atoms. Methods to functionalize NCD include for example: i) Direct chemical methods applied on NCD involving fluorination, organic free radical additions and fluorine displacement [15]; ii) Inducing hydrogen termination on the NCD for an example by exposing NCD surface with hydrogen atoms for 30 min at 700° C. [16]; iii) Electrochemical attachment schemes for binding of nitrophenyl linker to the H- or O-terminated diamond [1]; iv) Photochemical immobilization on H-terminated NCD [1; 16; 17]; v) Direct chemical reaction between NCD surface and radio-frequency plasma induced gas radicals [18; 19]. It has recently been shown that NCD can be used as an electrochemical biosensing surface [16]. The combination of its advantageous mechanical, chemical and physical properties makes NCD an ideal biosensor which is biocompatible and does not bio-degrade. The simultaneous broad band waveguiding properties, the intrinsic narrow band emission properties, the microfabrication and miniaturization properties, mechanical and chemical stability, surface functionalization properties, and biocompatibility makes NCD an attractive candidate material for remote, wireless, high-throughput in vivo diagnosis.
Of special interest is the surface and interface analysis made possible with ATR-IR where the solid IRE surface has been functionalized. In this manner the IRE is made an integral part of the measurement system. ATR-IR has been used in biosensing [11; 12], antibody recognition [20], in situ monitoring of bilayer formation [21], surface concentration determination [12; 20; 22], detection of protein conformational change upon adsorption or molecular interaction [23; 24], protein secondary structure determination [22; 25], and orientation in proteins and lipids [22; 26]. Recently some progress has also been made to functionalise IREs with crystals based on germanium [12; 20; 27] and silicon [11; 28]. An appropriately functionalised IRE can be used as a biosensor, for example, or for protein fishing. However, it is still a challenge to achieve a versatile sensing device based on ATR-FTIR spectroscopy. The major obstacle to overcome is to appropriately functionalise the IRE. Currently, commercially available IREs are not prepared for this; they are integrated within a complete ATR-FTIR accessory, which is mounted in a spectrometer. Due to their high cost, regeneration of the surfaces is of high priority. Moreover, when building up a functionalised IRE surface in a layer-by-layer fashion, for example, for immobilising peptides or other biomolecules, it is essential to optimise each step and test the functionality as well. Very recently, we have demonstrated a novel approach towards an in situ biosensing method based on ATR-FTIR spectroscopy with exchangeable functionalised sensor chips based on “upside-down” ATR measurements of ex situ prepared chips pressed onto a IRE by an anvil-type piston press [29]. None of these studies use NCD as detection or transducer material.
Mid-IR waveguides have attracted attention because of its use as remote chemical sensors or small volume samples chemical analysis. A mid-IR waveguides can be thought of as a miniaturized MRE wherein the light undergoes multiple internal reflections between media of different refractive indices and where the thickness or diameter of the waveguide is not too large in comparison with the wavelength of the propagating light. Mid-IR waveguides are characterized by multiple internal reflections that yield an interference pattern in the measured single beam intensity corresponding to the standing wave mode structure predicted by waveguide theory. Previous reports have shown the potential of surface enhanced infrared absorption in planar silver halide fibers [30], tapered mid-IR Ge elements [31; 32] and integrated Si optical waveguides [7; 8]. It is non-trivial to manufacture free-standing IR fiber waveguides and thus supported thin planar waveguides provide an interesting option to make IR waveguides for EWS applications. In contrast to the visible light region, where many suitable materials are commercially available and easy to manipulate, there are hitherto few options for appropriate IR-transmitting materials with high refractive index, low power attenuation, mechanical strength and chemical inertness. To the best of our knowledge we are only aware of three reports in the literature of mid-infrared planar waveguides. Two of them utilize monochromatic light [33], while the other uses broad band light focused into a hand-grinded 30 and 50 μm thick planar Ge waveguide employing a commercial IR microscope assembly [31]. None of these reports use diamond or NCD as waveguide material or as component in the waveguide.